The bleomycins (BLMs) are antineoplastic agents whose antitumor activity is recognized in the treatment of squamous cell carcinomas and malignant lymphomas (see, Umezawa, H. Antibiot. Chemother. 1978, 23, 76; Sikic et al., Eds. Bleomycin Chemotherapy; Academic Press: Orlando, Fla., 1985; Levi et al., J. Clin. Oncol. 1993, 11, 1300). The therapeutic effect of bleomycin analogues is believed to result from their selective oxidative cleavage of DNA (see, Stubbe et al., Chem. Rev. 1987, 87, 1107; Kane et al., Prog. Nucleic Acid Res. Mol. Biol. 1994, 49, 313; Claussen et al., Chem. Rev. 1999, 99, 2797; Hecht, S. M. J. Nat. Prod. 2000, 63, 158; and Chen et al., Nature Rev. 2005, 5, 102) and possibly also RNA (see, Carter et al., Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 9373; Holmes et al., Biochemistry 1993, 32, 4293; Hecht, S. M. Bioconjugate Chem. 1994, 5, 513; Abraham et al., Chem. Biol. 2003, 10, 45; and Tao et al., J. Am. Chem. Soc. 2006, 128, 14806). Much of the site-specific cleavage activity is thought to be effected by the N-terminal metal-binding domain, the C-terminal bithiazole region, and the linker domain (see, Carter et al., J. Biol. Chem. 1990, 265, 4193; Carter et al., Tetrahedron, 1991, 47, 2463; and Kane et al., J. Biol. Chem. 1994, 269, 10899). The least understood structural domain of BLM is the disaccharide moiety.
There is a carbamoyl group at the 3-position in the D-mannose moiety of the disaccharide that is thought to participate in coordination to a variety of metal ions (see, Oppenheimer et al., Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 5616). However, this does not appear to be the sole explanation for the presence of this sugar moiety as BLM and deglycoBLM (i.e., the BLM congener lacking the disaccharide) show similar cleavage efficacy in vitro (see, Ehrenfeld, G. M., Ph.D. Thesis, University of Virginia, 1986).
This ability of bleomycin to accumulate selectively on the surface or within tumor cells (see, Umezawa, H. Pure Appl. Chem. 1971, 28, 665) has been documented in numerous tumor imaging studies that utilized radionuclides bound to BLM (see, Jones et al., Med. Ped. Oncol. 1975, 1, 11; Silverstein et al., Cancer 1976, 37, 36; van de Poll et al., Nuclear-Medizin 1976, 15, 86; Rasker et al., Thorax 1976, 31, 641; Oyama et al., Radioisotopes 1976, 25, 567; Burton et al., Brit. J. Radiol. 1977, 50, 508; Tonami et al., Jap. J. Nucl. Med. 1977, 14, 217; Firusian et al., Strahlentherapie 1977, 153, 331; Bekerman et al., Radiology 1977, 123, 687; Stern et al., J. Nat. Cancer Inst. 1981, 66, 807; and Linder et al., J. Nat. Rev. Drug Discov. 2004, 3, 527). The innate tumor targeting nature of bleomycin is important in the design of synthetic analogues with improved properties. If the molecular basis of tumor targeting were understood it might also enable the selective delivery of other probes and drugs to tumor cells.
The tumor-specific behavior of bleomycin can be visualized by surface-conjugation of microbubbles. Microbubbles, usually consisting of a lipid-based shell encompassing an inert perfluorocarbon gas core (see, Rychak et al., J Control Rel 2006, 114, 288), were originally designed to improve the diagnostic ultrasound imaging of pathologic diseases within the human microvasculature (see, Hamilton et al., J. Am. Coll. Cardiol. 2004, 43, 453). While individual microbubbles may exist in varying sizes, they are usually smaller than red blood cells with a mean diameter typically within the range of 1-4 μm. With these inherent characteristics, microbubbles are generally regarded as pure intravascular tracers that travel freely within the (micro)circulation. Upon recognition of this behavior, there have been many efforts to direct microbubble attachment to specific cell types of interest by modifying the microbubble surface with different receptors, usually monoclonal antibodies. Initial studies have demonstrated that BLM tethered to a microbubble can adhere to tumor cells selectively (see, Chapuis et al., J. Am. Chem. Soc. 2009, 131, 2438).